The Citric Acid Cycle

Three main steps in the overall pathway (Citric acid)

1) Formation of acetyl-CoA from pyruvate

2) Oxidation of the remaining carbon atoms from pyruvate/acetyl-CoA to form both carbon dioxide and the further reduction of electron carriers.

3) Transport of these electrons and their use in oxidative phosphorylation.

Where does the citric cycle occur

The mitochondria

Membrane of mitochondria (task)

• The gradient of H+ ions (protons) is pumped to the intermembrane space (between the outer and inner membranes)

• These are then allowed back into the matrix to drive ATP production because of this physical gradient.

Fold of the inner membrane

Cristae:

• Increase the surface area for proton pumps and ATP synthase

How is ATP formed (simplfied)

• All of the electrons that have been made from both the citric acid cycle and from glycolysis are shunted through a few inner membrane proteins

• Eventually, these electrons are accepted by oxygen to form water molecules.

The gradient that we have formed allow for the formation of ATP through ATP synthase.

Overview of the Cycle (citiric cycle)

1) Acetyl-CoA attach to oxaloacetate to form citrate

2) Citrate loses a water molecule (temporarily), and regains it to form isocitrate

3) Isocitrate is oxidized to a-Ketoglutarate (+ NADH + CO2)

4) a-Ketoglutarate is further oxidized to succinyl-CoA. (+ NADH + CO2)

5) Succinyl-CoA is converted to succinate, losing its cofactor and producing energy in the form of GTP.

6) Succinate is converted to fumarate, producing reduced cofactors in the process (FADH2)

7) Fumarate is converted to malate (via hydration reaction)

8) Malate is converted back to the original oxaloacetate, repeating the cycle ( + NADH but NO CO2).

Major enzymes (convertion of pyruvate to acetyl-CoA)

1) Pyruvate dehydrogenase

2) Dihydrolipoamide Transacetylase

3) Dihydrolipoamide Dehydrogenase

Pyruvate dehydrogenase (Pyruvate to acetyl-CoA)(does what)

Decarboxylates the pyruvate to form a hydroxyl ethyl group, which then attaches to dehydrolipoamide transacetylase.

Dihydrolipoamide Transacetylase (pyruvate to acetyl-CoA)(does what)

Accepts the hydroxyl ethyl group, and transfers it to the acetyl coenzyme A group (Acetyl-CoA)

Dehydrolipoamide Dehydrogenase

Accepts the electrons from this process and, using different co-enzymes, transfers these to NAD+ to produce NADH.

Reaction 1 (acetyl-CoA to oxaloacetate)(Citric Acid Cycle)

• Removal of the acyl group from acetyl-CoA and reformation with oxaloacetate

• Enzyme: citrate synthase

Reaction 2 (Citrate to isocitrate)(Citric Acid Cycle)

Two reactions

• Removal of hydroxyl groups

• Rearrangement of these same groups

Enzyme: aconitase (lyase)

Form secondary alcohol from tertiary alcohol in two smaller steps.

Reaction 3 (Isocitrate to alpha-Ketoglutarate)

Two steps:

• Production of the reduced NADH and a apare proton (H+), which is then used in the next step.

• Oxalosuccinate is a somewhat unstable intermediate that loses its carboxylate group to form carbon dioxide (forming alpha-ketoglutarate).

Enzyme: isocitrate dehydrogenase.

Reaction 4 (alpha-Ketoglutarate to succinyl-CoA)

Three steps:

• The step produces additional fodder for oxidative phosphorylation later down the line.

• The last of the carbon molecules from the original process are now lost via carbon dioxide.

• Coenzyme A is required once again as an intermediary in the following step.

Enzyme: Alpha-Ketoglutarate dehydrogenase.

Gets NADH

Reaction 5 (Succinyl-CoA to Succinate)

• Removal of CoA (enzyme: succinyl-CoA synthetase)

• Indirectly produce ATP and produce GTP (GTP and ATP are interchangeable, where GTP is only required in smaller amounts for G-protein-linked reactions.

Reaction 6 (Succinate to fumarate)

• Enzyme: succinate dehydrogenase

• Making FADH2 is formed (another electron transporters)

• FAD is used rather than the co-factor NAD+, as FAD is a more powerful oxidizer and can help form the double bond required in fumarate.

Reaction 7 (Fumarate to Malate)

Enzyme: Fumarate hydratase (a lyase)

• Takes water and separates it into its constituent hydroxyl and proton, breaking the double bond found within fumarate.

Reaction 8 (Malate to oxalacetate)

Enzyme: malate dehydrogenase (another oxidoreduction reaction)

• Derive our last reduced co-factors, NADH, doen through the reformation of the double bond with oxygen.

formation of NADH

Citric Cycle

6CO2 + 10NADH/H+ + 2FADH2 + 4ATP

Regulation of the citric acid cycle (places)

1) Conversion of pyruvate to acetyl-CoA (most controlled)

2) Conversion of acetyl-CoA to citrate (Step 1)

3) Conversion of isocitrate to a-Ketoglutarate (Step 3)

4) Conversion of a-Ketoglutarate to succinyl-CoA (Step 4)

Acetyl-CoA (citric acid cycle activators/regulators)

• NADH and sucinyl-CoA downregulation

  • These are both substrates within the citric acid cycle, indication

Isocitrate (citric acid cycle activators/regulators)

• Upregulated via ADP and Ca+ downregulated via NADH and ATP

A-Ketoglutarate (citric acid cycle activators/regulators)

• Upregulated via Ca+, downregulated via succinyl-CoA and NADH.

Pyruvate Oxidation control (2 mechanisms)

• Feedback inhibition

• Phosphorylation control

Feedback inhibition (Pyruvate oxidation)

This occurs through the mechanism of acetyl-CoA and NADH, and functions as allosteris regulators of the 2 and 3 enzymes involved with pyruvate conversion:

• Dihydrolipoamide Transacetylase

• Dihydrolipoamide Dehydrogenase

Phosphorylation control (Pyruvate oxidation control)

• In order for E1 (pyruvate dehydrogenase) to function, it needs to have its phosphate removed

  • High levels of ATP will render the enzyme inactive

  • High levels of calcium, as well as magnesium, will stimulate the actions of the E1 enzyme.